Thymidylate synthetase (TSase) is an enzyme ubiquitous to all cells which is essential for the synthesis of DNA. Information on the detailed chemistry of the transformation of deoxyuridine monophosphate (dUMP) to thymidylate (T) is currently unavailable. The proposed research will produce these data. Furthermore, a thermodynamic description of both the quaternary structure of the enzyme and the reactions it catalyzes coupled with specific chemical modifications of the protein will yield information on the effect of these changes on the physico-chemical properties of the enzyme. This information will be useful in the understanding of the processes leading to uncontrolled cell replication. The physical chemistry of crystalline proteins has evolved to a point such that questions concerning the relation of structure and function may be asked and in many cases answers found. Thymidylate synthetase is an ideal model for asking thermodynamic questions concerning the structure/function relations. The enzyme contains two subunits and is thus a simple model for studying the equilibrium: iA1 yields (reversibly) Ai; (i equals 2). Furthermore, by the novel combination of ultracentrifugation and microcalorimetry, all state functions, delta G, delta H, delta S, and delta Cp for this reaction will be deduced. A combination of the above thermodynamic measurements with specific chemical modifications of the enzyme will lead to detailed information relating specific amino acids residues with the quaternary structure of TSase. Recent advances in theoretical descriptions of quaternary structure require precise determinations of delta G and delta H for critical evaluation. The proposed research will supply these data. Currently, very little information exists concerning the enthalpy changes accompanying enzymic catalysis. The varied chemical steps in the reaction dUMP plus MeTHF TSase yields T plus DHF make this an interesting reaction for calorimetric study. Furthermore, the metabolic significance of thymidine synthesis and its relevance to DNA synthesis stimulates our interest in these basic thermodynamic measurements.